Inhibition Assay Method and Device for Detection of Antibiotics

ABSTRACT

An aspect of the disclosure is a method and device for the detection of antibiotics in a sample. Embodiments include a microbial culture with growth indicators and sensitivity adjustment reagents. Adjustment reagents include a variety of antibiotic binders that can be added to microbial culture to reduce sensitivity to certain antibiotics. Some embodiments include multiple buffers with a variety of pKa ranges. Some embodiments also include a complete self-contained apparatus for sampling and testing.

REFERENCE TO PRIOR APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/578,935, filed May 9, 2006, which is based on and claims priorityfrom U.S. Provisional Patent Application No. 60/523,065, filed Nov. 18,2003, and U.S. Provisional Patent Application No. 60/574,252, filed May25, 2004, all of which are hereby incorporated by reference.

BACKGROUND

Antibiotics used in animal feed or to treat farm animals, such as dairyand beef cattle and swine, occasionally contaminate the food supply. Thehazards associated with these undesirable residues include allergicreactions, propagation of resistant organisms and other long-term healthrisks. As a result, government agencies such as the United StatesDepartment of Agriculture and the United States Food and DrugAdministration require monitoring of such residues in foods. Foods suchas milk, meat, poultry, seafood and animal feed are routinely tested forthe presence of such residues. We disclose herein a user-friendly methodfor detecting a variety of residues in a single, broad-spectrum test.

Various animal parts and excretions can be tested for the presence ofantibiotics. Tests for antibiotics in kidney include tests for hogkidney and veal bob kidney. Problems with some of the available tests,particularly in the pork industry, include sensitivities that do notmatch well with governmental or industry limits. Some tests areover-sensitive to certain antibiotics and under-sensitive to others.

Sensitivities of currently available microbial growth inhibition tests,are based primarily on parameters that affect test sensitivity to alldrugs. Such parameters include growth organism used, amount orconcentration of growth organism used, vessel dimensions, media volume,media type, mix of nutrients, incubation time and temperature. As aresult, if a test is adequately sensitive to some drugs, but overlysensitive to other drugs, other methods for sensitivity adjustment maybe needed.

Another problem with some tests is that they may require cumbersomeextraction procedures or use of organic solvents that must be removedfrom the sample prior to test operation. This adds a cumbersomeprocedural step. Some tests are also procedurally cumbersome, requiringincubation of a sample swab on a lawn of bacterial culture andinoculation of an agar surface with spores just prior to running thetest.

In addition to problems with slow testing time, over-sensitivity,under-sensitivity and extraction, none of the currently available testsare provided in an all-in-one format in which all test reagents andsampling devices are provided in one test instrument.

SUMMARY

We disclose herein various embodiments of a test apparatus and method todetect antibiotics in a test sample using growth inhibition of amicrobial culture. Some embodiments include all of the reagents for amicrobial culture in a ready-to-use format. The culture can include, forexample, nutrients, agar, one or more buffers, spores and colorindicator. Alternatively, any one or more of such components can beprovided separately, for example in a separate reagent compartmenthereinafter referred to as a “niblet”. In an embodiment of theready-to-use format, the user only has to add the sample, or sampleextract, and incubate for a prescribed time period, for example about1.5 to 4 hours, at a prescribed temperature, for example about 55-70degrees C., and observe the results.

If extraction is required, an extractant can be provided separately fromthe other reagents. In an example the extractant is in a niblet. Theniblet containing the extractant can be included in a test unitincluding other test components. The test unit can include the nibletcontained within a vial attached to the bottom of the test unit. Theseals of the niblet can be puncturable membrane seals such as metallicfoil seals or foil-like seals or plastic or plastic-like seals.Puncturing the seals allows the extractant to contact the culture. Theuser only has to add the sample to the extractant, contact the extractedsample with the culture, and incubate for the prescribed time period atthe prescribed temperature. The extractant can be a buffer solutioncapable of extracting antimicrobial drugs from a kidney sample, such asa bovine or porcine kidney sample. The extractant can also be a reagentfor merely removing the sample from a swab and transferring the sampleinto the culture.

Some embodiments include two or more buffers (hereinafter referred to asa polybuffer) having multiple pKa values. A polybuffer can improvepre-test and/or post-test stability of the culture and other aspects oftest performance. Post-test stability allows results to be retained atroom temperature for an extended period, for example 6-8 hours or more,without a change in test result. The polybuffer can be premixed withinthe culture or provided separately such as in a niblet for lateraddition.

An embodiment includes reducing sensitivity of the microbial culture tocertain inhibitors of culture growth. In some growth inhibition assays,the inhibition of microbial culture by antibiotics from a sample, orreversal of such inhibition, can be used as an antibiotic screeningmechanism. The sensitivity of such tests can be adjusted by contactingthe culture with at least one adjustment reagent. Examples of adjustmentreagents include binding protein, enzyme, chemical analogue and/orantibodies that bind to, or otherwise inhibit the effectiveness of,antibiotics. The adjustment reagent can be used to adjust thesensitivity of the test for at least one antibiotic or other culturegrowth inhibitor. An example of a microbial enzyme useful as anadjustment binder is an antibiotic receptor/binder from the cell wall ofmicrobes such as Bacillus (Geobacillus) Stearothermophilus (B.st.). Theadjustment reagent can be applied in a variety of ways, for examplecombined within a culture or broth, applied on top of a solid culture,such as an agar matrix, added on a sample swab or contained in adilution buffer.

Possible adjustment reagents include antibiotic receptors/bindersisolated from bacteria, monoclonal and polyclonal antibodies. When theadjustment reagent is an antibiotic binder, such as a binding protein,for example a bacterial antibiotic receptor or antibody, the adjustmentreagent can reduce the availability of antibiotics capable of inhibitingthe growth of the particular culture used, such as by binding to theantibiotic. Similarly, adjustment reagents can include substances that,rather than bind with the antibiotic, reduce the influence of theantibiotic on the growth organism.

Some embodiments include a test apparatus with a solid or semi-solidculture within a vial. The culture can include all or some of the testbiochemicals including agar, nutrients, color indicators, one or morebuffers and spores. In an embodiment, the culture, prior to the additionof test sample, is adjusted to a pH of greater than about 8. Such a pHcan help avoid culture contamination, for example with mold, andlengthen pre-testing shelf-life. The vial can include a sealed bottomend and a membrane seal over the top end. In test operation the user canpuncture the seal with, for example, a pipette tip or swab and thendispense the sample, for example a 200 microliter sample of milk, intothe vial.

Some embodiments include the complete test unit containing all of thereagents, premixed together and ready for use in a culture and, inaddition, a sampling instrument, such as a swab or probe and optionalextractant. The probe can be used to puncture the one or more sealsseparating the optional extractant from the culture. The probe can beused to absorb and apply the sample to the reagents within the testapparatus and can comprise an absorbent material such as an absorbent,fibrous, cotton-like or cotton material. An example of such a samplinginstrument is a swab or probe in the format of a POCKETSWAB (POCKETSWABis a registered trademark of Charm Sciences, Inc. Lawrence, Mass.). Theformat of the POCKETSWAB provides the advantage of controlled movementof the swab in a test device that provides physical support for theswab. For example, the swab can be controllably moved into the reagentcompartment containing an extraction buffer. Using the POCKETSWAB devicethe swab can remain in the extractant for the time desired. In additionto the convenience of delivering the swab to the customer in anall-in-one device, the POCKETSWAB provides the physical support so thatthe swab will remain within the extractant or buffer solution withoutthe user providing external support to the swab while soaking Aftersample extraction, the swab or swab tip is moved longitudinally througha seal thereby allowing the sample to flow onto or into the culture.

In a particular embodiment, the test method is used to reducesensitivity of a microbial culture to the beta-lactam family ofantibiotics. A beta-lactam binding protein is used to selectively reducetest sensitivity to the beta-lactams. Test sensitivity will be mostlyunaffected relative to antibiotics or inhibitors other thanbeta-lactams. In an embodiment, the beta-lactam binder is a receptorfrom B.st. and the culture is made of spores of B.st.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-9 are drawings depicting an embodiment in which the POCKETSWABformat is used.

FIG. 1 is an exploded perspective drawing of a swab 1 attached to a swabhandle 2. The swab 1 is used to obtain a sample.

FIG. 2 is an exploded perspective drawing of the whole POCKETSWABassembly 8 with the swab 1 in the pre-use position within the swab body7.

FIG. 3 is an exploded perspective showing the vial assembly 4 removedfrom the POCKETSWAB body 7.

FIG. 4 is an exploded perspective drawing of the vial assembly 4 showingcross-sectional lines 9 and niblet 5.

FIG. 5 is a cross-sectional view of the assembly shown in FIG. 4 withculture 15 at bottom of vial 4 below niblet 5 with extractant 11 orother additional reagent sealed therein.

FIG. 6 is an exploded perspective drawing of the in-use POCKETSWABassembly 8 in which the sample has been obtained and the swab 1 is movedthrough the seal 12 covering the vial assembly 4.

FIG. 7 is a cross-sectional view of the assembly shown in FIG. 6. Theswab tip 3 has broken through the seal 12 covering the vial assembly 4.The seal 13 on the top end of the niblet assembly 5 has not yet beenpunctured.

FIG. 8 is an exploded perspective drawing of the in-use POCKETSWABassembly 8 in which the sample has been obtained and the swab 1 is movedthrough the seal 12 covering the vial assembly 4 and the seal 13covering the top end of the niblet assembly 5 to contact the reagent 11within the niblet assembly 5.

FIG. 9 is a cross-sectional view of the assembly shown in FIG. 8. Theswab tip 3 has broken through the seal 12 covering both the vialassembly 4 and the seal 13 on the top end of the niblet assembly 5. Theseal 14 on the bottom end of the niblet assembly 5 has not yet beenpunctured.

FIG. 10 is a cross-sectional view of the vial assembly 4 after the swabtip 3 has broken through all of the membrane seals 12, 13 & 14—coveringthe vial assembly 4, the top and bottom ends of the niblet assembly 5.The liquid, for example extractant, having already been contacted withthe sample, is allowed to flow, or drip, into or onto the culture 15 atthe bottom of the vial assembly 4. After sample mixing and sealpuncturing the swab 1 is retracted back toward the pre-use position anddoes not contact the culture 15.

FIG. 11 and FIG. 12 are perspective drawings of an embodiment in whichmultiple vials are supplied for multiple samples. Perforation or otherdetachable attachments allow the user to determine the number of teststo run at one time. Within the bottom portion of each vial assembly 4 isthe culture 15. The optional niblet assembly 5 can also be provided. Atthe top of the vial assembly 4 there is a puncturable covering 12 whichcan be, for example, a puncturable membrane or foil seal. The vialassembly 4 can also be threaded 9 for optional attachment to thePOCKETSWAB test apparatus or for easy capping. In test operation a swabor pipette tip can be used to puncture the seal 12 on top of the vial 4prior to application of test sample to the culture 15. If the nibletassembly 5 is also provide within the vial assembly 4, a pipette tip canbe used to mix the sample with the reagents within the niblet assembly 5and to puncture the various seals.

DETAILED DESCRIPTION

An embodiment described herein involves a user-friendly method, deviceand kit for the detection of a broad range of residues of antibacterialcompounds in a sample such as an agricultural product. Antibiotics thatmay be detected include beta-lactams, sulfonamides, tetracyclines,macrolides, aminoglycosides, quinolones and amphenicols. In anembodiment, the user of the test adds the sample, incubates and observesthe results.

A useful mechanism of antibiotic detection is microbial growthinhibition. Examples of microbes useful in such an application include:B.st.; B. subtilis; B. megaterium; S. aureus; Ps. Aeuginosa; E. coli;and B. licheniformis. Generally microbes which exhibit detectable growthinhibition in the presence of antibiotics may be useful. Other examplesof microbial growth inhibition tests include those described in U.S.Pat. Nos. 5,354,663 and 5,489,532, the teachings of which areincorporated herein by this reference. Microbes, such as B.st., thatsporulate are particularly useful.

One of the benefits to microbial inhibition tests is that they can bebroad spectrum compared to family or antibiotic specific antibioticbinding based tests such as those utilizing antibodies or otherbacterial binders/receptors. For example, both beta-lactam andsulfonamide antibiotics will cause some inhibition of growth of B.st.The sensitivity of the currently available microbial inhibition testsare based on parameters including particular growth organism used,concentration of bacteria or spores, the nutrients provided and theincubation time and temperature used. Thus, in the example of B.st.,using one of those parameters to adjust test sensitivity may result inreduction of sensitivity to all or multiple of the antibiotics to bedetected. It may be desirable, however, to reduce test sensitivity of amicrobial growth inhibition assay to only certain antibiotics, or otherinhibitors such as only those for which the test is overly sensitive.For example, using B.st., it may be desirable to reduce test sensitivityto only beta-lactams and not sulfonamides or other drugs or inhibitors.

In an embodiment, a binding protein isolated from bacteria, sometimesreferred to as a receptor, antibiotic receptor, antibiotic binder orbacterial receptor, is utilized as an adjustment binder alone, or incombination with specific antibodies. Bacterial proteins that aresensitive to multiple antibiotics, such as a family of antibiotics, forexample the beta-lactams, can allow sensitivity adjustment of multipleantibiotics to which the particular binding protein is sensitive.Antibiotic binders, such as antibodies, that are more specific, allowmore specific sensitivity adjustment.

An antibiotic bound by such an adjustment reagent may be renderedunavailable, or less available, to inhibit bacterial growth. Althoughnot wishing to be constrained by theory antibiotic binders beingemployed on top of agar, or other solid culture, may create largemolecular weight substances that do not easily diffuse into a solidculture. Adjustment reagents, such as binders, combined into a solidculture or liquid medium, may inactivate, weaken or otherwise interferewith the antimicrobial properties or affect of the antibiotic. As such,substances other than antimicrobial binders may be useful to adjust testsensitivity including enzymes, such as beta-lactamase, that destroyantibiotic activity or otherwise inactivate antibiotics or substancesthat compete with antibiotics in the bacterial cell, such as analogues.One such substance is para-aminobenzoic acid (PABA), an analogue ofsulfonamides with which sulfonamides compete in certain bacterialmetabolic pathways. Similarly useful adjustment reagents may includesubstances that change the structure of an antibiotic or otherwisereduce the activity or make inactive the target antibiotic or antibioticfamily. Antibiotic binders may also include non-viable bacteria orbacterial extract such as cell wall extract.

Adjustment reagents, including antibiotic binders, can be eitherspecific to a particular drug for which the unadjusted test is overlysensitive, or have affinity to multiple drugs. The adjustment reagentmay be added with the sample or pre-mixed into a culture. One example ofan adjustment reagent for multiple drugs is a microbial receptor, suchas the beta-lactam receptor or receptors isolated from B.st. such asdescribed in U.S. Pat. Nos. 4,239,745 and 4,239,852 the teachings ofwhich are hereby incorporated by reference. Other possible sources ofbeta-lactam receptors include organisms from the genus Bacillusincluding B. subtilis, B. megaterium, and B. licheniformis. Otherpossible sources include S. aureus, Ps. Aeuginosa, B. licheniformis, andE. coli. Examples of useful specific adjustment reagents for particulardrugs include monoclonal or polyclonal antibodies. Other examples ofusing antibodies for sensitivity adjustment are provided in U.S. Pat.No. 6,319,466, the teachings of which are incorporated herein by thisreference.

Antibiotic binders can be isolated from bacteria and purified usingknown techniques. In an embodiment, beta-lactam binder isolated fromB.st. is used to adjust the sensitivity of a microbial inhibition assayusing B.st. as the growth organism. In such an embodiment, testsensitivity to all of the beta-lactams is reduced while test sensitivityto other antibiotics to which B.st. is sensitive remains relativelyunaffected. This is useful, for example, when target sensitivity isEuropean multi-residue levels (MRLs) and/or U.S. safe levels, both ofwhich regulatory levels allow beta-lactam in tissue that may be abovethe levels of detection of an unadjusted B.st. microbial growthinhibition assay.

Other antibiotic binders that may be useful include receptors isolatedfrom various part of bacterial or other cells including the ribosome orpart of ribosome to which certain antibiotics can bind. Examples ofantibiotics that bind to or otherwise inhibit ribosome function includetetracyclines, sulfonamides or fluoroquinolones. Bacterial growthinhibition by antibiotics such as tetracyclines, sulfonamides andfluoroquinolones may be adjusted by the addition of such binders. Inaddition, in some embodiments the adjustment reagent/binder can be anisolate from the same organism used to produce the culture.

Other useful methods of adjusting sensitivity include adding dead cellsand/or crude extract that contains the desired binders/receptors.

In one embodiment, depicted in the figures, a solid culture is providedin a reagent chamber such as a vial 4 or vial-like device. Located inthe same device can be a niblet 5 containing an appropriate extractantor liquid diluent 11. In an embodiment, an antibiotic binder is added tothe top of the solid culture 15. In another embodiment, an antibioticbinder is mixed into the solid culture 15. In a further embodiment, theantibiotic binder is stored separately and added to the solid culture15, along with the sample addition, prior to sample addition or soonthereafter. In another embodiment, the antibiotic binder is includedpre-applied to the sampling swab 1, for example by covalently binding tothe swab 1.

In an embodiment for detection of antibiotics in a sample requiringextraction, an extractant is used prior to adding the sample to theculture. An extractant that will separate antimicrobial drugs from asample, or from a swab or other sampling device, avoids complicatedextraction methods or use of organic solvents. An example of usefulextractants include a combination of Trizma Base and PotassiumPhosphate, such as Potassium Phosphate Monobasic in water at a pH ofabout 7 to about 8, for example approximately 7.5. Such an extractantbuffer can be provided separately from the culture or, for ease of use,in an all in one test device such as the POCKETSWAB format 8. Using thePOCKETSWAB format 8, after sample extraction the extractant (containingsample) is simply combined with the test reagents for incubation. Suchextractants may extract antibiotics from the sample or merely serve totransfer the sample, including antibiotic, from the swab or othersampling device into the culture.

The POCKETSWAB format 8 is described in U.S. Pat. No. 5,965,453, whichis incorporated herein by this reference. In the POCKETSWAB format 8,the extractant can be within a niblet 5. The niblet 5 can be situatedwithin the device, above the culture 15. The niblet 5 can be sealed onthe top 13 and bottom 14 ends such as with a membrane seal, for example,puncturable foil seals, on both ends to retain the buffer or extractanttherein. When using the POCKETSWAB format 8, the swab 1 is removed andcontacted with the sample. After sample contact, the swab 1 is used topuncture the first seal 12 (the vial 4 seal) and then the niblet topseal 12 thereby placing it within the niblet 5 and in contact with theextractant. After sufficient extraction time, the second sealed end 14(the bottom end) of the niblet 5 is punctured allowing the contents toflow onto the culture 15. The sample is incubated at above roomtemperature, for example between about 60 degrees C. to about 70 degreesC., such as about 64 degrees C. Spore germination and/or bacterialgrowth, or lack thereof, is determined by observation of changes ingrowth indicators within the culture 15.

Possible growth indicators include indicators that undergo a detectablechange as a function of the growth or inhibition of the culture, such aspH indicators and oxidation/reduction (redox) indicators. For example,indicators that change color in the presence of an acid or base such asto a yellowish color if the environment is acidic or purple/blue colorif basic or neutral. In the case of bacterial growth, or bacterialgrowth following germination of spores, acid is produced. Thus,indicators will reflect creation of an increasingly acid environment asbacterial growth proceeds. If bacterial growth or spore germination isinhibited, for example by antibiotics or other inhibitors in the sample,the environment will be less acidic. The color of the acid/baseindicator will reflect that relatively low acidity.

Some embodiments utilize a polybuffer. An example of such a polybufferis a combination of buffers, one with a pKa of above 7, for exampleabout 8 to about 11, and another with a pKa of below 7, for exampleabout 4.5 to about 6.3. The polybuffer can be included in the culture,in a separate niblet or provided separately for later addition. Whenpremixed in the culture, the polybuffer can be used to both stabilizethe reagent system prior to test operation and stabilize results afterthe testing is complete.

In a particular example of a polybuffer combined with the culture,borate and succinate are used. Borate helps provide a high pHenvironment to stabilize the culture during pre-test storage. Succinatehelps provide a low pH environment to stabilize the pH of the systemafter test operation. For example, in a negative sample the pH of theculture will be reduced as spores germinate and bacteria multiply. Aftertest completion the color of the culture will reflect the test results.The buffer with pKa of below 7, for example succinate, will helpstabilize the pH of the now acidic environment, thereby minimizing orpreventing further color change. External pressure for pH change, forexample decreased temperature after the test is removed from anincubator and the test returns to room temperature, may cause theculture to become more basic and, therefore, the test will appear morepositive. A buffer with pKa below 7 will help stabilize the result evenupon return to room temperature. In another embodiment Trizma Base isthe high pKa buffer and can be combined with a low pKa buffer such assuccinate.

Choice of buffers will be governed by a variety of factors. One factoris avoiding buffers that are particularly sensitive to the temperaturechanges within the test. Another factor is the starting pH of theparticular buffer. For example, Trizma Base may provide less temperaturestability as compared to borate. Trizma Base, however, can provide amore basic starting pH and, therefore, possibly better test sensitivityand stability as compared to borate. In an embodiment Trizma Base isused to adjust the pH of the pre-use culture to a pH of greater thanabout 8, for example about 10.5.

Adjusting the pH of the starting culture to a relatively high pH is alsoa method to improve test sensitivity and/or shelf life. A high startingpH, for example above about pH 7.5, for example about pH 8 or above,such as in the range of about pH 7.5 to about pH 11, may help preventpremature spore germination. The high pH environment may also help avoidmold contamination. Generally, the high pH environment may help extendthe shelf-life of the culture prior to use.

In another embodiment multiple test samples can be tested using a testplate for example a 96 well test plate. In such an embodiment mediaculture and adjustment binders can be provided together in the well. Inanother embodiment multiple vials are supplied, for example attached toeach other by a perforation or breakable plastic, so that one ormultiple tests can easily supplied to the user.

Example 1 Preparation of Solid Culture (Agar Matrix)

The following culture can be used for detection of antibiotics and otherinhibitors in a variety of matrices including, for example, urine, milk,water, poultry, seafood, feed and feed extracts and meat, such as kidneysamples.

A Bromocresol purple (BCP)/Tris Solution was prepared by combining 25 mLof a Trizma Base solution (TBS), the solution including 2.5 grams TrizmaBase in 100 mL of reverse osmosis/deionized water (RO/DI Water), with100 milligrams of BCP and mixing well. The media was prepared bydissolving 5 grams glucose and 1 gram Mueller Hinton Broth (MuellerHinton broth includes, in purified filtered water, 2 grams per liter(g/L) beef extract, 17.5 g/L casein hydrolysate acid and 1.5 g/L starch,pH 7.3 at 25 degrees C) into 100 mL RO/DI Water. Then a 0.01 mg/mLsolution of trimethoprim in RO/DI Water was prepared and 1.2 mL wasadded to the media. Next 20 mL of BCP/Tris (sterile filtered through0.45 micron filter) was added to the media.

Agar was prepared by combining 0.3 grams Difco Bacto-Agar (Item #0140-01), 0.225 NaCl and 17.485 RO/DI Water. The mixture was heated to95° C. and then removed from the heat and allowed to cool to 75° C. Next6 mL of media (prepared as described above with BCP/Tris) was added tothe agar and mixed for 5 minutes. The mixture was cooled to 57° C. and 1mL spore solution (concentration of 1 billion cfu/mL) was added andmixed together for 5 minutes. 0.200 mL was dispensed into the bottom ofa vial 4.

Example 2 Single Service Test

The dispensed vial 4 was prepared as described in example 1. Anextractant was prepared by adding 4.8 grams of 47.2% Trizma Base and52.5% Potassium Phosphate Monobasic to 1000 mL RO/DI Water (pH should be7.5+/−0.10). The extractant was sealed within the niblet 5 and theniblet was added to the vial 4. The vial 4 was heat sealed with foil.

Example 3 Single Service Kidney Swab Procedure

(Note: this procedure is described below using a format in which theswab is provided packaged within the test unit such as in thePocketSwab.)

The single service kidney swab procedure was run by first making a 3inch incision into kidney. The swab 1 was then withdrawn from the testunit, the test unit in the form of a POCKETSWAB, by gently pulling andtwisting the handle 2 out of the test unit body 7. The swab 1 was theninserted into the incision of the kidney and allowed to sit for 15minutes to allow full absorption of liquid into the swab 1. The swab 1was reinserted by gently pushing down and twisting to engage the threads6 and screwing the handle down slowly about halfway. The swab tip 3punctured the seals 12, 13 & 14 immersing the swab tip 3 into theextractant within the niblet 5 where it sat for 2 minutes. The swab 1was then screwed down all the way. After shaking and tapping the vial 4to get residual liquid into the bottom of the vial 4 and in contact withthe culture 15, we placed the vial 4 into a heat block set at 67° C. andincubated for 2.5 hours (if urine is tested instead of kidney, incubatefor 4 hours). After 2.5 hours the vial 4 was removed from the incubatorand vial color was observed in comparison to reference colors. If thenegative control did not match the color specified on the protocolincubation was continued for an additional 10 minutes.

Using the above procedure with the further addition of various amountsof beta-lactam receptor units to the top of the culture, from 0.3 unitsto 5.0 units, test sensitivity to penicillin G, was reduced by varyingamounts dependent on the amount of receptor added. See example 5 belowfor receptor unit determination.

Example 4 Single Service Urine Test

The culture is prepared as described in example 1. The extractant isprepared as described in example 2. The test is run the same as inexample 3 except that the swab tip 3 was allowed to sit in urine samplefor 10 seconds to allow full absorption into the swab tip 3 followed byincubation for 4 hours.

Example 5 Sensitivity Adjusted Single Service Kidney Test withAdjustment Binder (Receptor) Combined with Test Reagents

Culture is prepared as in Example 1. Prior to dispensing 200 microlitersinto the vial 4, antibiotic binder is prepared, in this example anantibiotic binder from the cell wall of B.st., also known as receptor.Varying amounts of inhibitory receptor units are mixed into the culture.Receptor is defined as protein removed from cellular membrane of B.st. Areceptor unit (1 U) is the amount of receptor that will, in the test,reverse the culture growth inhibition of 12.5 ppb of penicillin G.

Receptor is useful in a variety of forms including purified form, in theform of cell paste in which receptor is a component of the whole cell orin the form of broken cell fragments.

Results from experiments are shown in Tables 1, 2 and 3.

Results in Table 1 are from tests using the culture described in Example1 and with the addition of receptor added into rinse buffer (rinsebuffer composition is 4.8 grams of 47.2% Trizma Base and 52.5% PotassiumPhosphate Monobasic to 1000 mL RO/DI Water) at 0, 0.3 units (U), 1.0 U,2.0 U, 5.0 Upper test. Sample contained the following amounts ofpenicillin G: 0, 5 parts per billion (ppb), 10 ppb, 12.5 ppb, 25 ppb and50 ppb. As shown, 1.0 U was able to reverse the result of a 12.5 ppbpenicillin G sample. Results are either positive (+) or negative (−).

TABLE 1 Sample ID 0 5 ppb 10 ppb 12.5 ppb 25 ppb 50 ppb 0 U − + + + + +0.3 − − − + + + 1.0 − − − − + + 2.0 − − − − + + 5.0 − − − − + +

Table 2 data shows that when adding 1 U or receptor sensitivity to drugsother than beta-lactams results did not change. Results are eitherpositive (+), negative (−) or borderline between positive and negative(+/−). Abbreviations are penicillin G (PenG); sulfamethazine (SMZ);sulfadimethoxine (SDM); tylosin (TY); gentamicin (G); oxytetracycline(OT).

TABLE 2 Drug and Concentration w/out receptor With receptor Negative − −PenG 12.5 ppb + − PenG 25 ppb + + SMZ 50 ppb − − SMZ 100 ppb +/− +/− SMZ200 ppb +/− +/− SDM 50 ppb +/− +/− SDM 100 ppb +/− +/− SDM 200 ppb + +TY 300 +/− +/− TY 500 + + TY 800 + + G 400 +/− +/− G 600 +/− +/− G 800+/− +/− OT 250 + + OT 500 + + OT 750 + +

The results in Table 3 show that the receptor can be added to variouslocations or components within the test to achieve the same or similarresult. Results shown are from negative sample and samples containing12.5 ppb penicillin G and 50 ppb penicillin G. Results are eitherpositive (+) or negative (−).

TABLE 3 Receptor location 0 12.5 ppb 50 ppb Control - no − + + receptorReceptor added to − − + buffer Receptor added to − − + culture surfaceReceptor added − − + dried on swab Receptor mixed in − − +/− culture

Example 6 Preparation of Culture Media Containing Two Buffer

The following culture can be used for detection of antibiotics and otherinhibitors in a variety of matrices including, for example, urine, milkor kidney samples and can be used alone in a test container or in thesingle service test unit or in sensitivity adjustment examples.

BCP/Borate/Succinate Solution was prepared by adding 3.8 grams of Borateand 6 grams Succinate to 100 mL of reverse osmosis/deionized water(RO/DI Water) in 125 mL flask. 50 milligrams of BCP was added to a 50 mLconical tube and 5 mL of Borate/Succinate solution was added to the 50milligrams BCP and mixed.

To prepare media 5 grams glucose and 1 gram Mueller Hinton Broth wasdissolved in 100 mL RO/DI Water. 1.2 mL of a 0.01 mg/mL solution oftrimethoprim in RO/DI Water was prepared and added to the media. A 20 mLof BCP/Borate/Succinate was added to the media and the media was sterilefiltered through a 0.45 micron filter and then cooled to 4 degrees C.

Agar was prepared by combining 0.3 grams Difco Bacto-Agar (Item #0140-01), 0.225 NaCl and 17.485 RO/DI Water. The mixture was heated to95° C. and then removed from the heat and allowed to cool to 75° C. Next6 mL of media (prepared as described above with BCP/Tris) was added tothe agar and mixed for 5 minutes. The mixture was cooled to 57° C. and 1mL spore solution (concentration of 1 billion cfu/mL) was added andmixed together for 5 minutes. 0.200 mL was dispensed into the bottom ofeach of the vials.

After dispensing, tests a run test to determine if sensitivity isadequate. If sensitivity is not adequate the procedure is repeated usingeither more spores (under sensitivity) or less spores (ifoversensitive).

The following Table 4 shows results from antibiotic detection testsusing culture described in Example 6. In test operation 200 microlitersof various milk sample, spiked with known concentrations of antibiotics,were pipetted into test vials and incubated for the prescribed time (inthis test 2 hours 10 minutes) and prescribed temperature (64 degreesC.+/−2 degrees C.). Incubation was in the Charm I Inctronic incubator.Results were recorded immediately after test completion (Color 1) andafter being left at room temperature for 16 hours (Color 2). Resultslisted are in parts per billion (ppb). 4 samples were run at eachconcentrations except for raw negative milk (16 samples) and 3 ppb pen G(2 samples) and amoxicillin (2 samples). Abbreviations are penicillin G(PenG); sulfamethazine (SMZ); sulfadimethoxine (SDM); tylosin (TY);gentamicin (G); oxytetracycline (OT); pirlimycin (Pirl); amoxicillin(Amox); ampicillin (Amp); cloxacillin (Clox); ceftiofur sodium (Ceft);cephaprin (Ceph); tilmicosin (Til); sulfadoxine (SDN); neomycin (Neo).Color result 1 and 2 is a negative result, 3 is a borderline result and4 and 5 is a positive result.

TABLE 4 Color % Color % Tolerance Levels Drug: Conc 1 Pos 2 Pos USAEU/MRL PenG 4 5 100% 5 100%  5  4 PenG 3 5 100% 5 100%  5  4 Amox 6 5100% 5 100%  10  4 Amp 5 5 100% 4 100%  10  4 Clox 30 4 100% 5 100%  10 30 Clox 50 5 100% 5 100%  10  30 OT 200 2  0% 2  0% 300 100 OT 300 5100% 4 100% 300 100 SMZ 100 5 100% 5 100%  10 100 SMZ 200 5 100% 4 100% 10 100 SDM 25 4 100% 5 100%  10 100 SDM 50 5 100% 5 100%  10 100 G 3004 100% 4 100%  30 100 G 400 5 100% 5 100%  30 100 TY 40 5 100% 4 100% 50  50 TY 50 5 100% 5 100%  50  50 Pirl 100 4 100% 4 100% 400 100 Pirl200 5 100% 5 100% 400 100 Ceft 50 1  0% 1  0%  50*  100** Ceft 100 2  0%2  0%  50*  100** Ceph 10 5 100% 5 100%  20  60 SDN 100 5 100% 4 100% NLNL Til 80 5 100% 5 100% NL NL Neo 750 5 100% 5 100% NL NL  0% Raw 0 1*** 1  0% N/A N/A Milk: *parent drug listed **total parent andmetabolite ***individual bulk tanker samples

Example 7 Preparation of Culture Containing Two Buffer

The following culture can be used for detection of antibiotics and otherinhibitors in a variety of matrices including, for example, urine, meat,poultry, seafood, milk or kidney samples and can be used in a variety offormats including single service or sensitivity adjustment examples.

A Trizma/Succinate solution was prepared by adding 2.5 grams Trizma Baseand 6 grams Succinate to 100 mL of reverse osmosis/deionized water(RO/DI Water) in a 125 mL flask. 40 milligrams of BCP (see example 1 forBCP preparation) was then added to a 50 mL conical tube and 25 mL of theTrizma/Succinate solution was added to the 40 milligrams BCP and mixed.

The media was prepared by dissolving 5 grams glucose and 1 gram MuellerHinton Broth in 100 mL RO/DI Water. 1.2 mL of a 0.01 mg/mL solution oftrimethoprim in RO/DI water was added to the media. 20 mL of theBCP/Trizma/Succinate preparation was added to the media and the mediawas sterile filtered through a 0.45 micron filter and cooled to 4degrees C.

0.3 grams Difco Bacto-Agar (Item # 0140-01), 0.225 NaCl and 17 mL ofRO/DI water was combined and mixed and heated the mixture to 95° C. Themixture was removed from the heat and allowed it to cool to 75° C. andthen 6 mL media (with BCP/Tris) was added and mixed for 5 minutes. Themixture was cooled to 57° C. and 2 mL spore (concentration of 1 billioncfu/mL) was added and mixed for 5 minutes. 0.200 mL was dispensed intothe bottom of each vial.

After dispensing, tests were run to determine if sensitivity wasadequate. If sensitivity is not adequate the procedure is repeated usingeither more spores (if under sensitive) or less spores (ifoversensitive).

The following Table 5 shows results from antibiotic detection testsusing the culture described in Example 6. In test operation 200microliters of various milk samples, spiked with known concentrations ofantibiotics, were pipetted into vials and incubated for the prescribedtime (in this test 2 hours 30 minutes) and prescribed temperature (64degrees C.+/−2 degrees C.). Results were recorded immediately after testcompletion and 16 hours later. Incubation was in various Charm InctronicI incubators. Concentrations listed are in parts per billion (ppb). 4samples were run at each concentration except 16 negative samples wererun and 2 samples each for 3 ppb pen G and amoxicillin. Abbreviationsare pencillin G (PenG); sulfamethazine (SMZ); sulfadimethoxine (SDM);tylosin (TY); gentamicin (G); oxytetracycline (OT); pirlimycin (Pirl);amoxicillin (Amox); ampicillin (Amp); cloxacillin (Clox); ceftiofursodium (Ceft); cephaprin (Ceph); tilmicosin (Til); sulfadoxine (SDN);neomycin (Neo).

TABLE 5 Re- % 16 hr % Tolerance Levels Drug: Conc sult Pos Result PosUSA EU/MRL PenG 4 Pos 100% Pos 100%  5  4 PenG 3 Pos 100% Pos 100%  5  4Amox 6 Pos 100% Pos 100%  10  4 Amp 5 Pos 100% Pos 100%  10  4 Clox 30Pos 100% Pos 100%  10  30 Clox 50 Pos 100% Pos 100%  10  30 OT 200 Pos 0% Pos  0% 300 100 OT 300 Pos 100% Pos 100% 300 100 SMZ 100 Pos 100%Pos 100%  10 100 SMZ 200 Pos 100% Pos 100%  10 100 SDM 25 Pos 100% Pos100%  10 100 SDM 50 Pos 100% Pos 100%  10 100 G 300 Pos 100% Pos 100% 30 100 G 400 Pos 100% Pos 100%  30 100 TY 40 Pos 100% Pos 100%  50  50TY 50 Pos 100% Pos 100%  50  50 Pirl 100 Pos 100% Pos 100%  400 100 Pirl200 Pos 100% Pos 100%  400 100 Ceft 50 Pos 100% Pos 100%  50*  100**Ceft 100 Pos 100% Pos 100%  50*  100** Ceph 10 Pos 100% Pos 100%  20  60SDN 100 Pos 100% Pos 100% NL NL Tilm 80 Pos 100% Pos 100% NL NL Neo 750Pos 100% Pos 100% NL NL  0% Raw 0 Neg *** Neg  0% N/A N/A Milk: *parentdrug only **total parent and metabolite ***individual bulk tankersamples .

Example 8 Preparation of Culture Containing Two Buffer

The following culture can be used for detection of antibiotics and otherinhibitors in a variety of formats and matrices including, for example,urine, milk or kidney samples.

BCP/Borate/Succinate solution was prepared by adding 3.8 grams of Borateand 2.7 grams Succinate to 100 mL of reverse osmosis/deionized water(RO/DI Water) in a 125 ml, flask. 100 milligrams of BCP was added to a50 mL conical tube and 25 mL of the Borate/Succinate solution was addedto the 100 milligrams BCP and mixed.

To prepare the media 5 grams glucose was dissolved into 100 mL RO/DIwater. 1.2 mL of a 0.01 mg/mL solution of trimethoprim in RO/DI waterwas added to the media along with 20 mL of the BCP/Borate/Succinatepreparation. The media was sterile filtered through a 0.45 micron filterand cooled to 4 degrees C.

0.3 grams Difco Bacto-Agar (Item # 0140-01), 0.225 NaCl and 18 mL ofRO/DI water was combined and mixed and the mixture heated to 95° C. Themixture was then cooled to 75° C. after which 6 mL media was added andmixed for 5 minutes. The mixture was then cooled to 57° C. and 1 mLspore (concentration of 1 billion cfu/mL) was added and mixed in for 5minutes. 0.200 mL was dispensed into the bottom of each vial.

After dispensing, run tests to determine if sensitivity is adequate. Ifsensitivity is not adequate repeat procedure using either more spores(if undersensitive) or less spores (if oversensitive).

Example 9 Dispensing Culture

To efficiently dispense culture (such as is required in the previousexamples), containing spores of Bst, into the bottom of the vial 4, theagar must be heated to approximately 57 degrees C. Dispensing is donerapidly, for example in less than one hour, preferably in 45 minutes orless, so that the culture can be quickly cooled. If the culture is notquickly cooled, spores, for example B.st. spores, may germinateprematurely. That is, spores will germinate prior to application of thesample. Excess premature germination will reduce test sensitivity.

Another method for preventing premature spore germination, which can beused alone or in conjunction with rapid dispensing, is to increase thepH of the culture, for example in the range of about pH 7.5 to about pH11. The increase in pH provides non-optimal conditions for sporegermination. The increase in pH also allows more stability over longstorage times.

Example 10 Dilution Buffer

In some embodiments and examples, such as example 4, in which the samplematrix being tested is variable, a dilution buffer can be used tostandardize the sample matrix. In example 4, a test for urine wasdescribed. In this example, to standardize the urine samples prior totesting the sample is diluted approximately 1 to 20 with a mixture ofextractant (4.8 grams of 47.2% Trizma Base and 52.5% Potassium PhosphateMonobasic to 1000 mL RO/DI Water—pH should be 7.5+/−0.10) combined withabout 8-10 grams per liter beef extract. Other samples, including feedand water can be similarly diluted (for example water was diluted 1 to 5feed 1 to 30). By diluting the sample, test sensitivity is similarlyreduced.

1-10. (canceled)
 11. (canceled)
 12. (canceled)
 13. (canceled) 14-17.(canceled) 18-23. (canceled) 24-59. (canceled)
 60. A self-contained testapparatus for the microbial culture, growth inhibition, detection of anantibiotic in a test sample, said test apparatus comprising: a) a vialat a one end of the test apparatus, the vial containing a microbialculture, the culture comprising: (i) agar, (ii) spores, (iii) nutrients,and a (iv) pH indicator; b) a test apparatus body, the body attached toboth the vial and a cap, the body having a space in the center toreceive a sampler, the sampler attached to the cap and configured toobtain the sample and to transfer into the vial the sample to be testedfor antibiotics; c) a container, the container located within the vialand above the culture, the container comprising: (i) a cylinder having aone open end and an other opposite open end, (ii) a membrane seal overthe open ends to form a sealed compartment that is puncturable by thesampler, (iii) and an extraction reagent sealed within the sealedcompartment, wherein the sampler is used to obtain the sample and addthe sample to the extraction reagent and to the culture, and wherein thegrowth of the culture, or lack thereof, indicates the presence in thesample of an antibiotic.
 61. (canceled)
 62. The test apparatus of claim60 further comprising within the culture at least two buffers whereinone of said buffers has a pKa of above 7 and the other of said buffershas a pKa of below 7, wherein the pKa of above 7 allows pre-test storageand the pKa of below 7 allows post-test result stabilization.
 63. Thetest apparatus of claim 60 wherein the spores are spores of Bacillusstearothermophilus.
 64. The test apparatus of claim 60 furthercharacterized in that the antibiotic binding protein comprises a proteinisolated from Bacillus stearothermophilus.
 65. The test apparatus ofclaim 81 further characterized in that the antibiotic binding proteincomprises an antibody.
 66. The apparatus of claim 60 wherein theextraction reagent comprises a mixture of Trizma Base and PotassiumPhosphate Monobasic.
 67. The apparatus of claim 62 wherein one of saidbuffers is Trizma Base and the other of said buffers is succinate.68.-80. (canceled)
 81. The test apparatus of claim 60 wherein theculture further comprises an antibiotic binding protein and wherein theprotein reduces test sensitivity to an antibiotic that the proteinbinds.
 82. The test apparatus of claim 81 further characterized in thatthe antibiotic binding protein comprises a protein isolated fromBacillus stearothermophilus and wherein the spores are spores ofBacillus stearothermophilus.
 83. The method of claim 60 wherein the pHof the culture, prior to adding the sample is above about pH 8.0,wherein said pH of about 8.0 stabilizes the culture during pre-teststorage.
 84. The method of claim 60 wherein the pH of the culture, priorto adding the sample, is between about pH 7.5 and about pH 10.5, whereinsaid pH of between 7.5 and 10.5 stabilizes the culture during pre-teststorage.
 85. The apparatus of claim 60 wherein the sample comprises akidney sample.
 86. The apparatus of claim 60 wherein the samplecomprises a tissue sample.
 87. The apparatus of claim 60 wherein thesample comprises a urine sample.
 88. The apparatus of claim 60 whereinthe sample comprises a feed sample.
 89. The apparatus of claim 81wherein the antibiotic binding protein reduces test sensitivity to thepenicillin type beta-lactams.
 90. The apparatus of claim 60 wherein thetest sampler comprises a swab.
 91. The apparatus of claim 60 wherein theextraction reagent comprises a solution that includes a Trizma Base. 92.The apparatus of claim 60 wherein the extraction reagent comprises asolution that includes a Potassium Phosphate.
 93. The apparatus of claim60 wherein the extraction reagent comprises a solution that includes aPotassium Phosphate Monobasic.
 94. The apparatus of claim 60 wherein theextraction reagent comprises a solution of a pH between about 7.0 toabout 8.0.